Characterization of primitive hematopoietic cells in normal human peripheral blood.
Characterization of primitive hematopoietic cells in normal human peripheral blood.
复制标题
正常人外周血中原始造血细胞的表征。
作者：
C. Udomsakdi;P. Lansdorp;D. Hogge;DS Reid;A. Eaves;C. Eaves
通过将正常人骨髓细胞接种在感受态贴壁细胞饲养层上启动的5周龄长期培养物（LTC）中存在的克隆原性细胞总数允许定量更原始的造血输入前体细胞类型，称为LTC起始细胞（LTC-IC）。以前的研究表明，LTC-IC也循环，因为当来自正常血液的低密度（< 1.077 g/mL）、T细胞耗尽部分的细胞维持在LTC培养基中的经辐照的骨髓来源的饲养层上时，克隆形成细胞的产生持续许多周。我们现在表明，5周后，在这种重建的LTC中存在的克隆形成细胞的数量与外周血（PB）细胞的输入数量在很宽的细胞浓度范围内线性相关，从而允许通过有限稀释分析定量循环LTC-IC。使用这种方法，我们发现正常成人循环中LTC-IC的浓度为2.9 +/- 0.5/mL。这比循环克隆形成细胞（即爆发形成单位-红系细胞+集落形成单位[CFU]粒细胞-巨噬细胞+CFU-粒细胞、红系细胞、单核细胞、巨核细胞）的浓度低约75倍，并且代表相对于所有有核细胞的LTC-IC频率比正常骨髓穿刺样本中测量的频率低约100倍。表征研究表明，大多数循环LTC-IC是小的（低前向光散射和侧向散射），CD 34+，Rh-123暗淡，HLA-DR-和4-氢过氧环磷酰胺耐药细胞，与正常骨髓中的LTC-IC的分化和增殖潜力难以区分。分离正常血液的低密度、T细胞耗竭、CD 34+和HLA-DR（低）或Rh-123（暗）组分，得到高度富集的细胞群，其为0.5%至1% LTC-IC（超过低密度、T细胞耗竭步骤约1，500倍富集），纯度与迄今报道的最富集的人骨髓LTC-IC群相当。然而，基于这些性质的PB LTC-IC的纯化不允许它们与相同样品中的相当大比例（> 30%）的克隆形成细胞物理分离，这与先前对骨髓中的LTC-IC和克隆形成细胞的发现相反。这些研究表明，在正常成人的血液中存在一个相对较小但易于检测的功能定义的原始造血细胞群体，这些细胞与骨髓LTC-IC（一种被认为具有体内重建潜力的细胞类型）具有相同的特性。（400字处截断摘要）
The total number of clonogenic cells present in 5-week-old long-term cultures (LTC) initiated by seeding normal human marrow cells on competent adherent cell feeder layers allows for the quantitation of a more primitive hematopoietic input precursor cell type referred to as an LTC-initiating cell (LTC-IC). Previous studies have suggested that LTC-IC also circulate because production of clonogenic cells continues for many weeks when cells from the light-density (< 1.077 g/mL), T-cell-depleted fraction of normal blood are maintained on irradiated, marrow-derived feeder layers in LTC medium. We now show that the number of clonogenic cells present in such reconstructed LTC after 5 weeks is linearly related to the input number of peripheral blood (PB) cells over a wide range of cell concentrations, thereby permitting the quantitation of circulating LTC-IC by limiting dilution analysis. Using this approach, we have found the concentration of LTC-IC in the circulation of normal adults to be 2.9 +/- 0.5/mL. This is approximately 75-fold lower than the concentration of circulating clonogenic cells (ie, burst-forming units-erythroid plus colony-forming units [CFU] granulocyte-macrophage plus CFU-granulocyte, erythroid, monocyte, megakaryocyte) and represents a frequency of LTC-IC relative to all nucleated cells that is approximately 100-fold lower than that measured in normal marrow aspirate samples. Characterization studies showed most circulating LTC-IC to be small (low forward light scatter and side scatter), CD34+, Rh-123dull, HLA-DR-, and 4-hydroperoxycyclophosphamide-resistant cells, with differentiative and proliferative potentialities indistinguishable from LTC-IC in normal marrow. Isolation of the light-density, T-cell-depleted, CD34+, and either HLA-DR(low) or Rh-123(dull) fraction of normal blood yielded a highly enriched population of cells that were 0.5% to 1% LTC-IC (approximately 1,500-fold enriched beyond the light-density, T-cell-depletion step), a purity comparable to the most enriched populations of human marrow LTC-IC reported to date. However, purification of PB LTC-IC on the basis of these properties did not allow them to be physically separated from a substantial proportion (> 30%) of the clonogenic cells in the same samples, in contrast to previous findings for LTC-IC and clonogenic cells in marrow. These studies show the presence in the blood of normal adults of a relatively small but readily detectable population of functionally defined, primitive hematopoietic cells that share properties with marrow LTC-IC, a cell type thought to have in vivo reconstituting potential.(ABSTRACT TRUNCATED AT 400 WORDS)